The development of pulmonary hypertension (PH) involves altered vascular smooth muscle cell (VSMC) proliferation. However, the mechanisms are still unresolved. Previous data have shown the remodeling in PH is induced, at least in part, by increased levels of endothelin-1 (ET-1) and alterations in the expression of ET receptors such that ETA receptors are increased and ETB receptors are diminished. Our lab has previously shown that up-regulated ETA signaling stimulates reactive oxygen species (ROS) generation and VSMC proliferation. Previous studies have shown that ROS-mediated oxidation of the epidermal growth factor receptor (EGFR) induces its covalent dimerization. This oxidized dimeric EGFR is thought to be irreversibly cross-linked and constitutively active. Conversely, nitric oxide (NO) bioavailability is diminished in PH potentially decreasing peroxynitrite mediated tyrosine nitration. This may be important as the nitration of EGFR known to inhibit its activity. We hypothesize that the mechanism by which EGFR nitration is regulated in VSMC is via ETB mediated activation of NOS1 located in the VSMC layer. Further we hypothesize that the loss of ETB signaling leads to enhanced ROS signaling through ETA leading to EGFR activation. To test these hypotheses in Aim 1 we will use VSMC to determine if ET-1 stimulates VSMC growth through ROS-mediated covalent dimerization and activation of EGFR and if antioxidants and EGFR antagonists reduce this effect. Next, we will over-express ETB receptors to determine if this will enhance EGFR nitration, prevent ROS- mediated activation of EGFR, and lead to VSMC growth arrest. To confirm the importance of aberrant EGFR signaling in the development of PH in Aim 2 we will utilize an in vivo model of PH induced by monocrotaline injection in the rat. We will determine if the covalent dimerization of EGFR is increased in PH and EGFR nitration is decreased. Next we will determine if EGFR antagonists will prevent the VSMC proliferation and vessel remodeling associated with the development of PH. Finally, we will determine if using gene delivery of the ETB receptor to the rat lung using electroporation will increase EGFR protein nitration, reduce VSMC proliferation, and alleviate the changes associated with progression of PH. PUBLIC HEALTH RELEVANCE: Pulmonary hypertension (PH) is a rare but severe disease mostly affecting young women and ultimately leading to death. The central pathological event of PH is uncontrolled growth of pulmonary vascular smooth muscle cells (VSMC) resulting in further disease progression. This application aimed to delineate the role of posttranslational epidermal growth factor receptor (EGFR) modifications in VSMC growth and PH development and suggests the therapy for EGFR inhibition based on the novel method of selective intra-VSMC gene electroporation.